High throughput system for performing assays using electrochemiluminescence  including a consumable shaking apparatus

ABSTRACT

The present invention relates to a system for performing assays on a solid phase to measure the level of analyte in a sample. Such a system may perform immunoassays using electrochemiluminescence (ECL) including a counterbalanced orbital shaking apparatus for assay consumables. The counter-balanced orbital shaking apparatus also incubates the assay consumables, and has a cooling system to maintain a preset temperature within the shaking apparatus.

CROSS-REFERENCE TO RELATED APPLICATION

The present international application claims priority to U.S.provisional patent application No. 62/673,371 filed on May 18, 2018,which relates to international application No. PCT/US2016/026242 filedon Apr. 6, 2016, which claims priority to a provisional applicationentitled “Consumable Shaking Apparatus” filed on Apr. 6, 2015 bearingSer. No. 62/143,557, and to another provisional patent applicationentitled “Throughput System for Performing Assays UsingElectrochemiluminescence Including a Consumable Shaking Apparatus” filedon Mar. 22, 2016 bearing Ser. No. 62/311,752, which are all incorporatedherein by reference in their entireties.

The present international patent application describes an improvement tothe high throughput system described and claimed in international patentapplication No. PCT/US2016/026242.

FIELD OF THE INVENTION

The present invention is directed to a system for performingimmunoassays using electrochemiluminescence (ECL).

BACKGROUND OF THE INVENTION

The use of binding assays on a solid phase is a common approach tomeasuring the levels of analytes in a sample. There are many types ofnatural and synthetic binding reagents (for example, antibodies, nucleicacids, aptamers, receptors, ligands, etc.), solid phases (e.g., thesurface of a container or well or the surface of a microparticle) andassay formats (direct binding, sandwich, competitive, etc.) that areknown in the art of solid phase binding assays. One specific examplethat illustrates the types of processing steps that are typical for asolid phase binding assay is a sandwich immunoassay which uses twoantibodies directed against the target analyte, one of which isimmobilized on a solid phase and the other carrying a label that isdetectable through some detection technique (e.g., using fluorescence,chemiluminescence, electrochemiluminescence, absorbance, or themeasurement of an enzymatic activity). When the solid phase is thesurface of a well in a multi-well plate, typical steps in this formatmay include: (i) adding a sample to a well and incubating to allowanalyte in the sample to be captured by the immobilized antibody in thewell; (ii) adding the labeled detection antibody to the well andincubating so that the detection antibody binds to captured analyte toform a labeled “sandwich” complex on the solid phase and (iii) measuringthe labels that are present in sandwich complexes on the solid phase.

Optionally, the wells may be washed before or after any of the steps toremove any unbound materials prior to addition of new solutions. Duringthe incubation steps, the plates may be shaken to reduce the time andimprove the reproducibility of the binding reactions. One exemplarydetection technology that may be used to measure labels during themeasuring step is electrochemiluminescence (ECL) detection, whichemploys labels such as derivatives of ruthenium tris-bipyridine thatemit light when in proximity to oxidizing or reducing electrodes underappropriate chemical conditions (see, e.g., U.S. Pat. No. 6,808,939which is incorporated herein by reference in its entirety).Instrumentation and consumables that are designed to carry out bindingassays in multi-well format with ECL, detection have been described(see, e.g., U.S. Pat. No. 7,842,246 which is incorporated herein byreference in its entirety). The '246 patent describes multi-wellconsumables having integrated electrodes within the well that are usedas solid phase supports for antibodies or arrays of antibodies. Theformation of labeled complexes on the electrodes is measured by applyinga voltage to the electrodes and measuring the resultant ECL signal. AnECL read buffer, such as a buffer containing tripropylamine or anothertertiary amine (see, e.g., U.S. Pat. No. 6,919,173 which is incorporatedherein by reference in its entirety) may be added to the well prior toapplying the voltage to provide chemical conditions that lead toefficient generation of ECL. A number of alternative protocols forcarrying out ECL assays have also been described including protocolswith an additional step during which capture antibodies are immobilizedfrom solution (see, e.g., US Published Patent Application No.20140256588 which is incorporated herein by reference in its entirety)and protocols where the measurement step includes an amplification stepprior to the ECL measurement (see, e.g., US Published Patent ApplicationNo. 20140272939 which is incorporated herein by reference in itsentirety).

In certain situations, ECL electrodes or other solid phases may betreated with a material (a “blocker” or “blocking reagent”) thatprevents non-specific binding of analytes or assay reagents. Thistreatment may be carried out as a separate “blocking” step or blockingreagents may be included in the buffers or diluents used during othersteps of an assay procedure. Examples of useful blocking reagentsinclude proteins (e.g., serum albumins and immunoglobins), nucleicacids, polyethylene oxides, polypropylene oxides, block copolymers ofpolyethylene oxide and polypropylene oxide, polyethylene imines anddetergents or surfactants (e.g., classes of non-ionicdetergents/surfactants known by the trade names of Brij, Triton, Tween,Thesit, Lubrol, Genapol, Pluronic, Tetronic, F108, and Span),

Heretofore, the steps in ECL immunoassays are completed by variousindividual machines. For example, the washing of the multi-well platesis accomplished by plate washing machines; the pipetting of samples andreagents into multi-well plates is carried out by mechanized pipettingmachines having a large number of pipette tips; the stirring of thesamples and antibodies is carried out by mechanical shakers; and theexcitation of analyte-antibody complexes and sensing of the emittedlight are conducted by plate reading machines. However, there remains aneed in the art for an overall system that integrates all theseindividual machines into a single interconnected system that improvesefficiency, provides the ability to clean multiple pipette tips during arun, and provides thermal control to satisfy the operating temperatureranges of the reagents and/or the samples.

SUMMARY OF THE INVENTION

An aspect of the present invention relates to an ECL immunoassay systemcomprising a housing that encloses a pipette dispenser, a plurality ofmulti-well trays adapted to hold ECL complexes attached to electrodescontained in the tray, an incubator, an ECL reader and a cooler. Theincubator can be a counterbalanced assay consumable shaking apparatusdescribed in international application No. PCT/US2016/026242, which ispublished as WO 2016/1644777 and which is previously incorporated byreference in its entirety. The cooler is located proximate to a backsurface of the housing and the cooler can comprise one or morethermoelectric coolers. This aspect of the present invention is directedto a novel way of cooling the internal space of the ECL immunoassaysystem generated by various components such as the incubator orconsumable shaking apparatus.

The present invention is directed to an ECL immunoassay systemcomprising a housing that encloses a pipette dispenser, a plurality ofmulti-well plates adapted to hold ECL complexes attached to electrodescontained in the plurality of multi-well plates, which are sized anddimensioned to be stored in an incubator, a blower, an ECL reader and atleast one cooler, The at least one cooler is located proximate to a backsurface of the housing, and the incubator comprises at least one airflowchannel disposed therewithin. The blower is positioned to pull air fromthe housing through the at least one airflow channel, and the air isthen ducted to an inlet of the at least one cooler to cool said air.

The ECL immunoassay system may also has a temperature sensor connectedto the at least at least one cooler, and the temperature sensor ispositioned proximate to an entrance of the at least one airflow channel.The temperature sensor acts as a thermostat of the at least one coolerand can be a thermistor. Preferably, the at least one cooler comprises athermoelectric cooler.

The air is ducted to an inlet of the at least one cooler through a hoodpositioned above the blower and the hood is connected to a chimney.Thereafter, said air is blown by the at least one cooler through aplenum back into the housing.

The at least one airflow channel in the incubator comprises asubstantially horizontal airflow channel and a substantially verticalairflow channel. The incubator may also provide shaking motion to stirthe plurality of multi-well plates.

BRIEF DESCRIPTION OF THE FIGURES

In the accompanying drawings, which form a part of the specification andare to be read in conjunction therewith and in which like referencenumerals are used to indicate like parts in the various views:

FIG. 1 is a diagram showing an exemplary redox ECL reaction in animmunoassay;

FIG. 2 is a front view of the inventive ECL immunoassay system;

FIG. 3 is a top view of the system of FIG. 2;

FIG. 4 is a perspective view of the pipette tip washing manifolds;

FIG. 5 is a cross-sectional view of a number of chimneys in the washingmanifolds of FIG. 4 with an exemplary pipette tip;

FIG. 6 is a close-up view of the washing manifolds of FIG. 4;

FIG. 7 is a perspective view of the enclosure of the system of FIGS. 2and 3 with elements omitted for clarity showing a flow plenum;

FIG. 8 is a close-up view of a flow diverting plate shown in FIG. 7;

FIGS. 9(a)-(b) are a flow charts of exemplary methods for operating theinventive system;

FIGS. 10(a)-(b) are perspective front views showing detailed views ofthe shaker apparatus with portions of the housing omitted to show theinternal mechanisms; FIG. 10(c) is an expanded view of the top eccentricmount and the counterbalance shown in FIG. 10(b); and FIG. 10(d) is atop view of the driving mechanism and the bottom eccentric mounts;

FIGS. 11(a)-(i) show detailed views of the storage assembly includingvarious alternative configurations of sets of vertically aligned storageunits within the storage assembly (FIGS. 11(c)-(i)).

FIGS. 12(a)-(b) show two alternative configurations of sets ofvertically aligned storage units within the storage assembly andcounterbalance placements within the storage assembly relative to thestorage unit sets.

FIGS. 13(a)-(d) show one embodiment of a latching mechanism used in thestorage units of the apparatus, where FIG. 13(b) is a partial view of amicrotitre plate.

FIGS. 14(a)-(b) show an embodiment of the shaker apparatus with aninternal air flow path.

FIG. 14(c) is a side view of the shaker apparatus with the inlet airflow to the shaker apparatus and the outlet airflow from the blowerassembly labeled by arrows. FIG. 14(d) is a partial rear view and FIG.14(e) is a partial front view of the inventive ECL immunoassay systemwith certain components omitted for clarity showing an improved coolingconfiguration.

DETAILED DESCRIPTION OF VARIOUS EMBODIMENTS

Unless otherwise defined herein, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. The terms“tray” and “plate” are used interchangeably herein. The term “hotel” and“shelve(s)” are also used interchangeably herein. Further, unlessotherwise required by context, singular terms shall include pluralitiesand plural terms shall include the singular. The articles “a” and “an”are used herein to refer to one or to more than one (i.e., to at leastone) of the grammatical object of the article. By way of example, “anelement” means one element or more than one element.

One embodiment of the present invention is directed to system 10 asshown in FIGS. 2 and 3. System 10 advantageously handles all the stepsof the solid phase binding assays (e.g., ECL immunoassays) describedabove, and preferably is a fully automated system that can handle anumber of multi-well plates 4. The system, as described below, isespecially well suited for carrying out ECL-based solid phase bindingassays, however, one of average skill in the art could adapt the systemto run other assay formats and/or detection technologies using similarprocessing steps by appropriate selection of the plate reader componentand the assay consumables and reagents. All the necessary and optionalmachineries are contained within housing or enclosure 12, as well assamples, reagents, buffers, washing and cleaning liquids, electronicsand waste storages. Preferably, enclosure 12 is supported by castorwheels to render system 10 mobile.

As best shown in FIG. 2, enclosure 12 has upper compartment 14 and lowercompartment 16. Upper compartment 14 houses the machineries, samples andreagents. Lower compartment 16 stores electronics 18, which may includea computer, interfaces to control the machineries and to receive dataand communication from the machineries, input/output devices including agraphical user interface for technicians and other users to select theproper protocol from a list of predetermined protocols and a WiFi forremote communication with other users. Lower compartment 16 also storescontainers 20 to store washing liquids including deionized water with orwithout surfactants or soaps and store waste or used water, describedfurther below.

Upper compartment 14 houses a number of equipment and machineriesmounted on floor or deck 15, including hut not limited to shelves 22,also known as hotel 22, sized and dimensioned to store a plurality oftrays that contain samples or reagents or that will be used in the assayprocess, bar code reader 24 (not shown) located below shelves 22, androbot arm 26 designed to deliver the trays and their lids (if available)to other machineries and return them to shelves 22. Upper compartment 14also contains shaker and incubator 28, plate washer 30 designed to washunattached materials from the wells of multi-well assay plates, multiplechannel pipettor 32 for delivering liquids to the wells of multi-wellassay plates and, pipette tip washing manifold 34 to clean the pipettetips from pipettor 32 after each use. Upper compartment 14 also housestray/plate reader 36 and thermoelectric coolers 38, which are solidstate coolers. Plate reader 36 is, preferably, an ECL plate reader forcarrying out measurements based on the ECL reaction depicted in FIG. 1.Alternatively, other types of plate readers (such as absorbance,fluorescence or chemiluminescence-based plate readers) could be used tocarry out assays using other detection approaches. The coolers may havethe same wattage capacity, e.g., 200 W. Alternatively, one of morecoolers—preferably the ones that are located near a heat source such asplate reader 36, may have higher capacity, e.g., 250 W.

Also mounted on deck 15 are pipetting deck 40, which in this embodimentcontains four spaces sized and dimensioned to retain four trays, andgantry 42, which supports multichannel pipettor 32 for movement alongdeck 15 in three-dimensions. Preferably, pipette tip washing manifold 34comprises two manifolds 34 a and 34 b, each contains a number ofchimneys corresponding to the number of pipette tips being used.Preferably, one manifold contains water with a small amount ofsurfactant, such as those discussed above in connection with theblocking reagents, and the other manifold contains deionized water,described further below.

Before assay system 10 is started, the plates that will be used in theassay process are loaded onto shelves 22. Any multi-well plates can beused so long as the plates are sized and dimensioned to work with themachineries. Preferably, the shape and dimensions of the plates are inconformance with established standards for assay plates—such as thoseset by the Society for Laboratory Automation and Screening (SLAS)—andthe plate-processing components (such as the plate washer, pipettor,reader, robot arm, etc.) are configured to process plates meeting thesame standards. To enable high-throughput parallel processing ofmultiple samples, the assay plates 4 used for carrying out assayreactions are preferably multi-well plates, Preferably, the number andarrangement of wells follows an established standard such as the24-well, 96-well, 384-well and 1536-well formats (most preferably, the96-well plate format), although any well arrangement is possible, Forcarrying out ECL-based assays, assay plates as described in U.S. Pat.No. 7,842,246 may be used. The highest throughput can be achieved byusing components that simultaneously process all the wells in a platesimultaneously. For example, for processing 96-well assay plates, system10 preferably comprises a 96-channel pipettor and 96-channel platewasher.

In addition to the assay plates, other plates may be loaded that providesamples to be tested or reagents used in the assays such as assaydiluents, detection reagents, read buffer (e.r., TPA solution), blockingagents, and tip washing reagents bleach solution). These plates may havea multi-well format, preferably with the same well density as the assayplates, The use of multi-well plates is advantageous when it isdesirable to transfer different samples or reagents into the differentwells of an assay plate. Reagents or samples that will be transferred toall the wells of a plate can also be provided in multi-well plates, orthey can be provided in plates having a single large well (i.e., areagent reservoir). Advantageously, the wells of the plates used forsamples and/or reagents may be conical or round-bottom wells to reducedead volume. The plates may be sized so that the volume of liquid issufficient for only one assay plate, or for multiple assay plates (e.g.,through the use of higher volume deep well plates. Plates that store thesamples and reagents are positioned in predetermined locations onshelves 22. These positions are preselected when defining the protocolso that robot arm 26 knows where to extract and return the plates orreservoirs. Robot arm 26 is controlled by the computer stored in lowercompartment 16, which also contains the software to operate system 10.

To start system 10, an operator selects a protocol among a list ofprotocols to be executed. Protocols are chosen based in part on theassay format to be performed. Illustrative assay formats are describedbelow. Robot arm 26 preferably checks whether the plates and reservoirsare located where they are supposed to be and whether certain plates,such as the plates/reservoirs containing the samples or antibodyreagents, have a lid to minimize evaporation. Preferably, all plates andreservoirs have bottom surfaces of substantially the sane size, so thatall can fit snugly on tray pipette deck 40. The operating computer wouldnotify the operator if a tray or reservoir is mis-located or is missinga lid. Each plate 4 or reservoir placed on shelves 22 preferably has abar code, as best shown in FIG. 1. Bar code reader 24 can read these barcodes and communicate to the operating computer whether the correct trayor reservoir is loaded on shelves 22. After this check, robot arm 26would extract one or more trays, e.g., an assay plate 4, a sample platecontaining samples to be tested and/or a reagent plate (for example, aplate containing a diluent, blocker or a detection reagents such asantibodies labeled with ECL, labels), and place the plates on platepipetting deck 40. Optionally, the assay plate may be transferred to theplate washer and washed prior to placement on deck 40.

Thereafter, multichannel pipettor 34 may perform a number of preselectedactions according to the selected protocol. In one possible protocol,pipettor 34 uses 96 pipette tips to aspirate detection reagents from thewells of the reagent plate and inject a defined volume of the same intothe wells of an assay plate with immobilized antibodies on the surfaceof each well (for example, an ECL assay plate with antibodiesimmobilized on an electrode in each well). The pipettor then uses asimilar process to transfer a defined volume of each sample from thewells of the sample plate to the wells of the assay plate. Robot arm 26can put the assay plate with the samples and detection reagents intoshaker/incubator 28 to mix the samples and detection reagents whileincubating same. During the period when the plate is incubating,additional plates may be processed in series using the same set ofoperations.

After the samples and detection reagents are fully incubated, robot arm26 removes the tray from shaker/incubator 28 and brings it to the platewasher 30. Plate washer 30 has a pair of tubes for each well. One tubeinjects a washing liquid from a container 20 stored in lower compartment16 into the well and the other tube aspirate the same well and discardsthe used liquid into a waste container 20 also stored in lowercompartment 16. Preferably, the elevation of the aspiration tube islower than that of the injection tube. As discussed above, one purposeof washing plate 4 is to remove any analyte or detection reagent that isunattached to the well, as well as any components of the sample thatcould interfere with the assay measurement.

While the plate is on the washer, robot arm 26 transfers a reagentreservoir containing a read buffer from shelves 22 to deck 40. After theplate is washed, robot arm 26 carries the washed plate to deck 40, wherepipettor 34 transfers read buffer from the reagent reservoir to theplate. Robot arm 26 then transfers the plate to reader 36 where theassay measurement is carried out (e.g., in the case of an ECLmeasurement, by reader 36 applying a voltage to the electrodes in thewells to initiate the ECL reaction described above). The results areobtained by reader 36, and transferred to the operating computer storedin lower compartment 16. After completion, robot arm 16 returns theplates to shelves 14.

It is noted that the present invention is not limited to the stepsdescribed above. System 10 can execute any protocol involving any numberof steps in any sequences involving the machineries and equipmentdescribed above.

According to the selected protocol, pipettor 32 may be used to carry outmultiple pipetting steps on each of multiple plates in a run. Aninventive aspect of the present invention is the use of pipettorsemploying disposable pipette tips, where the tips are cleaned betweencertain operations within a run and replaced at a lower frequency suchas between runs. Additional inventive aspects relate to the specificcleaning procedures, reagents and subsystems used to clean tips thathave been selected to maintain a high processing throughput while alsoproviding negligible cross-contamination of samples. In one embodimentof the invention, pipette tips are washed between each cycle ofoperations (as defined below) carried out on an assay plate, to preventcross-contamination of the wells in different assay plates. In someprotocols, it may also be advantageous to wash the pipette tips betweenoperations in one cycle, especially when the sequence of pipetting stepsprovides the possibility of cross-contaminating sample or reagentplates. Thus, types of carryover include sample carryover and reagentcarryover. The tip-cleaning processes of the invention enabletip-cleaning to be carried out in less than 90 seconds (preferably, lessthan 60 seconds) while achieving an effective carry-over of less than 10ppm, preferably less than 1 ppm or less than 0.1 ppm and preferably lessthan 0.01 ppm or 0.001 ppm, where the effective carry-over is the amountof a solution 1 transferred into a solution 2 after the two solutionsare pipetted as numbered (1 then 2) using the same pipette tip.Effective carry-over may be determined, for example, by comparison of atest assay condition (using washed, reused pipette tips) to controlassay conditions using fresh (unused) pipette tips for each sample. Thecontrol conditions may include running a Control Sample 1 in which adefined quantity of solution 1 is spiked into solution 2. The controlconditions may also include running a Control Sample 2 that is solution2, but unspiked with solution 1. The results for solution 2 under thetest assay condition are compared to the difference in assay signals,e.g., analyte concentration, between Controls Sample 1 and ControlSample 2 under the control condition to determine the effectivecarry-over. See also Weibel et al., J. Lab. Automation 15:369-378(2010). One of ordinary skill in the art understands how to adaptmethods for measuring carryover to different assay platforms andautomated systems.

In one embodiment of the invention, the carryover of a protein analytefrom a first sample to the following sample pipetted using the same tipis less than 1 ppm, preferably, when the analyte is measured byimmunoassay. In another embodiment of the invention, the carryover of anucleic analyte from a first sample to the following sample pipettedusing the same tip is less than 1 ppm when analyzed by a nucleic acidhybridization or amplification assay.

To minimize effective carryover, the tip washing procedure preferablycomprises: (i) one or more washing steps that physically removematerials on a tip that could lead to inaccurate assay results and (ii)an inactivation step in which the tip is exposed to an inactivationcondition or reagent (e.g., solution) that inactivates any of suchmaterials so as to reduce or eliminate the ability of these materials toaffect assay results even if they are not fully removed by the washingsteps. The inactivation step could include treatment of the tip withheat, with electromagnetic radiation (e.g., the use of UV light toinactivate nucleic acids in samples or reagent) and/or with a gaseous orliquid chemical reactants that react with materials that could causecarryover effects the use of chemical oxidants such as bleach orhydrogen peroxide, acids or bases such as HCl or NaOH solutions,cross-linking agents such as formaldehyde and/or alkylating agents suchas ethylene oxide))). Preferably, the inactivation step comprisestreatment of the pipette tip with a bleach solution. These conditionsand reagents significantly reduce effective carryover of protein ornucleic acid analytes when pipetting a series of samples using the samedisposable pipette tip. Using the tip washing procedures of theinvention, disposable tips may be used to process 20 or more samples,preferably 100 or more samples, before requiring replacement with freshtips.

One embodiment of the tip washing procedures of the invention uses thefollowing process. To clean the pipette tips 33, robot arm 26 removes aninactivation reagent reservoir (e.g., a reservoir containing a bleachsolution as in the description below) from shelves 22 and places itsecurely on plate pipette deck 40. Referring to FIGS. 4-6, pipette tipwashing manifold 34 preferably has two manifolds for physically washingthe pipette tips. First manifold 34 a preferably uses a mixture of waterand surfactants to rinse the pipette tips and second manifold 34 bpreferably uses deionized water. Both manifolds have a plurality ofchimneys 44 that match the number of pipette tips 33 on pipettor 32.Pipettor 32 is supported by gantry 42 and is movable in three directionsin order to move along deck 15. In one embodiment, pipettor 32 ispositioned over either manifold 34 a or 34 b and is positioned so thatpipette tips 33 are located between chimneys 44, in other words pipettetips 33 are mis-aligned with the openings of chimneys 44. Next, anyremaining contents inside pipette tips 33 are discharged on to themanifold without going into and contaminating chimneys 44. Thereafter,pipettor 32 is moved to a position above the bleach solution reservoirand is inserted into the bleach solution. A first volume of bleachsolution is aspirated into pipette tips. Preferably, this first volumeis larger than the volume of any prior sample or reagent(s) so that thebleach solution migrates a sufficient height inside pipette tip 33 tooverlap a previous height of sample or reagent(s), The bleach solutionis then expelled from pipette tips 33. A bleach solution can be reused anumber of times, e.g., 10 times, until a fresh bleach solution reservoiris needed. Optionally, the steps associated with treatment with bleach(i.e., the inactivation steps) may be omitted for pipetting steps wherethe effect of carryover on the assay is likely to be small.

Thereafter, pipettor 32 is moved to a position above first manifold 34 aand pipette tips 33 are aligned directly over chimneys 44. As best shownin FIG. 5, pipettor 32 dips pipette tips 33 into chimneys 44 butmaintains a gap 46 between tip 33 and chimney 44. Washing liquid with anamount of surfactant from a container located below in lower compartment16 is pumped into chimneys 44 from the bottom through conduit 48 andmanifold 50 to be distributed to chimneys 44. Optionally, a flowrestrictor 51 is positioned upstream of each chimney 44 to ensureuniform fluid flow into gap 46 from manifold 50 below. A flow restrictorcan be a section of reduced diameter. A second volume of water andsurfactant is aspirated into pipette tip 34, wherein this second volumeis larger than the first volume. Additional water and surfactant ispumped through gap 46 to wash the outside of pipette tips 33. Tomaximize this outer flow of water, gap 46 preferably has a constantclearance. In other words, the outer shape or surface of pipette tip 33matches the inner surface of chimney 44 to maintain a constant clearancebetween tip 33 and chimney 44. Preferably, this clearance is between0.25 mm and 1 mm, more preferably between 0.5 mm and 0.75 mm.

After being washed with the surfactant solution in first manifold 34 a,pipettor 32 moves pipette tips 33 to second manifold 34 b and the samerinsing is repeated but with deionized water. A third volume ofdeionized water is aspirated into pipette tips 33, wherein the thirdvolume is greater than the second volume. In one example, the firstvolume is about 75 ml, the second volume is about 100 ml and the thirdvolume is about 125 ml.

To clean pipette tips 33, both the inside and the outside of the pipettetips should be cleansed. For internal washing, the amount of aspiratedvolume at each washing step should be progressively larger withprogressively “cleaner” solution, i.e., closer to clean water. Forexample, in the discussion above, the aspirated volumes progressivelyincreases from the first volume to the third volume and from a bleachsolution to a soapy solution (with surfactant) to deionized water.Alternatively, the bleach solution may be omitted, The washing isseparated into at least two reservoirs (34 a, 34 b). A rough wash inreservoir 34 a and a fine wash in reservoir 34 b. Within each reservoir,contaminants are effectively removed iterative wash cycles, i.e.,preferably using a directional flow. For external washing, pipette tips33 are located proximate narrow gaps 46 to get more shear force from theflow from manifold 50. Flow restrictions 51 may be positioned upstreamof gap to control and increase the flow through gaps 46.

This washing process allows disposable pipette tips to be re-used insystem 10. Fresh disposable pipette tips are installed onto pipettor 32at the beginning of each run, and can be used throughout the run and aredisposed at the conclusion of a run.

Referring to FIG. 6, first and second manifold 34 a and 34 b, preferablyhas level sensor 52 positioned on a wall thereof. In one embodiment,level sensor 52 is an optical reflectivity sensor that emits an IR(infrared) beam toward a transparent window 54, which preferably isacrylic. The index of refraction of window 54 is close to that of thewash liquid but is different than that of air, When air is behind window54, the difference between the indices of refraction between window 54and air is sufficiently high to cause a higher amount of IR to reflectby window 54. When washing liquid is behind window 54, the difference ofindices of refraction between window 54 and washing liquid issufficiently similar so that more IR is transmitted through window 54.Sensor 52 is capable of detecting higher IR transmission indicating thatthe liquid level is at window 54. This would signal to the operatingcomputer to shut down the pump to stop the flow of washing liquid, untilthe liquid is drained through drain holes 56. Drain holes 56 areconnected to a waste container located in lower compartment 16.

Advantageously, level sensor 52 can be used to establish a constant filllevel within pipette tip washing manifold 34. The pump can be shut offand drain holes 56 can be pinched when sensor 52 senses that the levelreaches window 54. This fill level is known to the operating computer,and in the event that droplets of waste are hanging off of pipette tips33, pipettor 32 can position pipette tips 33 away from chimneys 4.4 andlower tips 33 to an elevation above the fill level but sufficient forthe drops of waste to touch the liquid. This allows the waste dropletsto be transferred to the liquid in washing manifold 34 without touchingthe pipette tips to this liquid, which may have been previously used towash pipette tips 33 and may contain contaminants.

According to another aspect of the present invention, an advantage ofenclosing the machineries inside enclosure 12 is that the temperatureand/or humidity inside enclosure 12 can be controlled and theevaporation of reagents and other liquids can be minimized. Enclosure 12does not need to be sealed from the environment; however, the inside ofupper compartment 14 does not actively exchanging air with outsideenvironment. Upper compartment 14 is enclosed by a top surface, sidesurfaces, back surface and deck 15. The front surface comprises one ormore sliding or hinged doors. In certain applications, it is desirableto maintain the temperature within upper compartment 14 between about23° C. and about 27° C. and within ±1° C. at certain selected presettemperature within this temperature range.

Referring to FIG. 7, another inventive aspect of the present inventionrelates to the controlled airflow within upper compartment 14. Since anumber of machineries and other objects are present on deck 15, they canobstruct the air flow and redirect the air flow in an uncontrolledfashion. Thermoelectric coolers 38 generally take in air inside uppercompartment 14 horizontally at about their center, cool/warm the air anddischarge air vertically at their top and their bottom, FIG. 7illustrates enclosure 12 with the machineries and other componentsomitted for clarity. The top surface of enclosure 12, shown at referencenumber 58 in FIG. 2, and deck 15 also omitted for clarity. Below topsurface 58 of enclosure 12, a second top surface 60 is positioned belowtop surface 58 to create a flow plenum 62 at the top of uppercompartment 14. Preferably, second top surface 60 is spaced at asufficient distance from top surface 58 to allow the discharged air toflow through. The dimensions of flow plenum 62 can be adjusted smallerto speed up the air flow or larger to slow it down. Second top surface60 has at least one ingress 64 located proximate to the top discharge ofthermoelectric cooler 38, and at least one egress 66 near the front ofupper compartment 14. As shown, the top discharged air enters flowplenum 62 at ingress 64 and flows along the plenum until it reachesegress 66 near the front of upper compartment and is forced to flowdownward to modulate the temperature of the machineries before flowingback into the thermoelectric cooler at its intake. Without flow plenum62, the flow pattern from the top discharged air may not reach the frontportion of upper compartment 14, because the discharged air may bouncedoff of top surface 58 toward the horizontal intake without travelling tothe front of upper compartment 14.

Additionally, an inclined flow diverter 68 as shown in FIGS. 7 and 8 ispositioned directly below the bottom discharge of thermoelectric coolers38 to divert the flow along deck 15 to direct the air flow toward thefront of upper compartment 14 and then upward and back toward thethermoelectric coolers' horizontal intake. In another embodiment, asecond flow plenum 62 can be provided with deck 15. Whereby a bottomsurface is positioned below deck 15 and ingress 64 and egress 66 areprovided on deck 15.

The modified air flows at the top and/or at the bottom of uppercompartment 14 result in longer airflow paths from the top and bottomdischarges of thermoelectric coolers 38 back to their horizontal centerintakes. The longer airflow paths provide more efficient distribution ofairflow throughout the upper compartment and reduce temperature gradientwithin the enclosure and to maintain the temperature difference withinupper compartment 14 to be within +1° C.

Protocols

ECL immunoassay system 10 is capable of performing any number ofassaying protocols. Preferably, the assay protocol for processing eachplate is broken down into a series of timed processing cycles of equalduration, where each cycle involves the processing of a single plate ondeck 15 and the different cycles carried out on individual plates may beseparated by plate incubation periods. This approach can provideextremely high-throughput processing, while maintaining precise controlof the timing of assay steps and greatly simplifying the scheduling ofindividual automated operations. As long as each cycle has a duration ofN minutes (which means the operations or steps within a cycle take lessthan N minutes) and the incubation time between any two adjacent cyclesfor a given plate is at least Y minutes, then system 10 can batch Y/Nplates in a run without having to access two plates at the same timewhile maintaining consistent timing for all the assay processing andincubation steps on all the plates.

In one embodiment of this “timed cycle” approach, the individual cyclesthat make up the processing sequence for an assay protocol, are createdby modifying a generic multistep cycle by omitting steps that are notrequired in that specific cycle and, for steps involving fluidtransfers, by specifying the number of volumes of the transfers. Themodified cycles are achievable within the time duration of the fullgeneric cycle and do not require any modification to the overallscheduling of cycles.

An exemplary flow chart of one generic cycle is illustrated in FIG.9(a), as are some of the opportunities for modifying the cycle toproduce assay specific cycles. In step 70, a target plate is selected.In step 72, a decision whether to wash the plate is made. If YES, thenin step 74, a wash protocol is selected and a wash buffer is selected.After step 74 or if the decision in plate washing from step 72 is NO,then the protocol proceeds to step 76, where reagent (or sample) isadded. A reagent (or sample) source and a reagent (or sample) volume areselected. Thereafter, another decision whether to add a second reagent(or sample) is made in step 78. If YES, then in step 80, another reagent(or sample) source and volume are selected. After step 80 or if thedecision from step 78 is NO, then the protocol proceeds to step 82 wherethe plate is incubated and stirred. An incubation time and an incubationlocation, e.g., shaker 28 or shelves 22, are selected. Next, in step 84,a decision whether to wash the pipette tips is made. If YES, anotherdecision whether to wash with a inactivating solution such as bleach ornot is made at step 86. After step 86 or if the decision from step 84 isNO, then the protocol proceeds to step 88 and place the plate on reader36.

In one example, one assay protocol may have the following cycles,created by modifying the generic cycle of FIG. 9(a), with the followingsteps/operations:

Cycle 1. Pull assay plate and blocking reagent reservoir from shelves22, add blocking reagents to plate using pipettor 32 and put plate inshaker 28.

Cycle 2. Pull assay plate from shaker 28 and sample plate from shelves22, wash assay plate at plate washer 30, add samples to plate usingpipettor 32, put plate on shaker 28.

Cycle 3. Pull assay plate from shaker 28 and detection reagent reservoirfrom shelves 22, wash assay plate, add detection reagent to plate usingpipettor 32, put plate on shaker 28.

Cycle 4. Pull assay plate from shaker 28 and read buffer reservoir fromshelves 22, wash assay plate, add read buffer to plate using pipettor32, place plate of reader 36 for analysis.

In this example, each cycle takes 3 minutes or less to run, and if theincubation time on shaker 28 is 60 minutes, then system 10 can runbatches of 20 multi-well plates without interference between plates.

In another example, the system may be used to run protocols that includean incubation that is short or comparable in duration to the duration ofa cycle, e.g., incubation times in the range of 10 seconds to sixminutes. This protocol is suitable for running assays for high abundanceof analytes with short incubation instead of requiring dilutions. Forincubations that are short relative to the length of a cycle, theincubation may be carried out as a step within the cycle, In this case,the plate may be left on the deck (either without shaking duringincubation or using the pipettor to mix through up and down pipetting)or the plate may be transferred to the shaker, incubated and transferredhack to the deck within the time frame of a single cycle. In the casewhere the incubation time is a multiple M of the cycle time N (i.e., theincubation time=M×N), an interleaved process can be used thatinterleaves the pre-incubation processing steps (comprised within apre-incubation subcycle of duration A) and the post-incubationprocessing steps (comprised within a post-incubation subcycle withduration B), where A+B=N (the total time for an individual cycle). Inthis case, a processing cycle for the interleaved process may comprise(i) process a plate in the batch using the pre-incubation subcycle or,if no plate is available (e.g., all the plates have already undergonethe pre-incubation subcycle) then idle for time A and (ii) process aplate in the batch that has completed the M×N time incubation using thepost-incubation subcycle or, if no plate is available (e.g., no plateshave completed the M×N incubation) then idle for time B. Using thisinterleaved approach, it is possible to continuously process plates andthere is no upper limit in batch size. If the assay process comprises anadditional long incubation step of time Y, as described above for thetimed cycle approach, then the length of the long incubation step willdetermine that batch size that can be run during the protocol.

In yet another example, system 10 can be operated as illustrated in FIG.9(b). In step 90, system 10 is initiated, wherein the consumables suchas plates, reservoirs, pipette tips, etc. are loaded, as describedabove. In step 92, a protocol is selected by the operator. In step 94, afirst single multi-well assay plate is processed, using a firstprocessing cycle that includes one or more of exemplary processing steps(a)-(f) are performed.

a. removing a single multi-well tray from a shelve,

b. optionally, washing said single multi-well tray,

c. depositing a sample to be tested into the wells on said singlemulti-well tray,

d. depositing at least one reagent into the wells on said singlemulti-well tray,

e. optionally, washing said single multi-well tray to remove remaininganalytes;

f. placing the optionally washed single multi-well tray in theincubator,

The first processing cycle repeated with additional single multi-wellassay trays until the incubator is full as indicated in step 96, i. e ,(h) repeating steps (a)-(f) with another single multi-well tray untilthe incubator is full. The period of incubation in this example is thesum of the time to fill the incubator with multi-well trays. After theincubator is filled with processed trays, step (g), as illustrated instep 96, the first tray, which is now fully incubated, is removed and,optionally, processed using a second processing cycle 94 that includesone or more of steps (a)-(f). The second processing cycle, if used, isthen repeated with additional single multi-well trays until theincubator is full. Similarly, additional processing cycles 94 may alsobe carried out on the batch of plates as needed for a specific assayprotocol. The final processing cycle will also comprise processing step(h) (shown as step 98 in FIG. 9b ) in which the assay tray istransferred to a plate reader (e.g., an ECL tray reader) for analysis.The final cycle is repeated until all assay trays are placed in thereader in step 99 and analyzed. The number of multi-well trays stored inthe incubator is equal to the incubation period divided by the time tocomplete the longest of the processing cycles (i.e., steps (a)-(f) and,in the final cycle, (h)).

The method illustrated in FIG. 9(b) can be modified by first determiningthe number of multi-well trays that can be processed and stored in theincubator during the incubation period, and then processing the traysaccording to processing steps (a)-(f). The system can process theremaining trays, and after the first tray is fully incubated the traysare moved to the reader for ECL analysis on a first-in-first-out basis.

Any number of protocols can be designed based on the teachings herein bythose of ordinary skill in the art. The present invention is not limitedto any particular protocol.

Descriptions of System 10's Components

The machineries and equipment shown and described above, andparticularly in FIGS. 2 and 3 can be specifically designed or can becommercially purchased. Shelves 22 are preferably custom built for theapplications intended. Bar code reader 24 can be a commercialoff-the-shelf component. Robot arm 26 can also be a commercialoff-the-shelf component. Plate washer 30 can also be a commercialoff-the-shelf component, and is available from Biotek, Inc. Reader 36can also be a commercial off-the-shelf component, and is available fromMeso Scale Diagnostics, Inc. as MESO QuickPlex SQ 120 Reader. ThisReader is described and claimed in commonly-owned pre-grant U.S. patentapplication publication no. US2014/0191109, which is incorporated hereinby reference in its entirety. Multichannel pipettor 32 andthermoelectric coolers 38 are also commercial off-the-shelf components.Pipette washing manifolds 34 can be specifically built, or purchased andmodified to improve the washing effectiveness. Gantry 42 is preferablyspecifically built for system 10.

Shaker 28 can be a commercial off-the-shelf component; however, in theembodiment of system 10 discussed above, shaker 28 is inventive anddescribed and claimed in commonly owned provisional application entitled“Consumable Shaking Apparatus” filed on Apr. 6, 2015 bearing Ser. No.62/143,557, which is incorporated herein by reference in its entirety.Relevant portions of this earlier provisional application are reproducedbelow.

A shaker and incubation apparatus 28 is shown in FIGS. 10(a)-10(d). Theapparatus includes an orbital. shaker assembly (101), including ahorizontal orbiting platform (102), and an assay consumable storageassembly (103) positioned on the platform (102). The storage assembly(103) includes a shelving subassembly (104) and a counterweight (105)positioned within the storage assembly at a height or plane thatsubstantially corresponds to the center of mass of the orbitingcomponents of the apparatus, i.e., the storage assembly and the orbitingplatform. The shelving subassembly includes a plurality of sets ofvertically aligned storage units. The apparatus depicted in FIGS.10(a)-1(b) includes four sets of vertically aligned storage units(106-109). Each storage unit (110) is sized to accommodate an assayconsumable (111) and includes a latching mechanism (112) to secure theconsumable within the storage unit and to ensure that each consumablepositioned within the subassembly is subjected to the same orbitalshaking momentum, velocity, and direction.

Examples of assay consumables suitable for use with the inventioninclude, but are not limited to, vials, flasks, beakers, assaycartridges and cassettes, microtitre plates, e.g., multi-well plates,slides, assay chips, lateral flow devices (e.g., strip tests),flow-through devices (e,g., dot blots), solid phase supports forbiological reagents and the like. In certain embodiments, test sites inthe assay consumable are defined by compartments in the assayconsumable, e.g., wells, chambers, channels, flow cells and the like, Ina specific embodiment, the assay consumable is a microtitre plate, e.g.,comprising 6, 24, 96, 384 or 1536-wells. More particularly, the assayconsumable is a 96-well microtitre plate.

Referring to FIGS. 10(a) and 10(d), the orbital shaker assembly 101includes a rotating shaft (113) that extends from the orbital shakerassembly (101) into the assay consumable storage assembly (103) in thevertical Z-axis. The counterweight (105) is operatively connected toshaft (113) at or near the centroid plane or the plane that includes thecenter of mass of the assay consumable storage assembly (103). Topeccentric (115) is operatively connected to the top of the rotatingshaft (113) and to a chassis or surface of assay consumable storageassembly (103), discussed further below.

Referring to FIG. 10(d), the orbital shaker assembly (101) has drivingmotor (121) connected to rotating shafts (113), (123) and (125) by meansof a belt (127). Preferably, the belt (127) is grooved or is a timingbelt. One or more pulleys (129) are positioned to ensure that the shaftsare driven and are driven at substantially the same rotational speed.Shafts (123 and 125) are operatively connected to first bottom eccentric(131) and second bottom eccentric (133). Bottom eccentrics (131, 133)are operatively connected to horizontal orbiting platform (102) whichsupports assembly (103) or operatively connected directly to assembly(103), and as stated above top eccentric (115) is operatively connectedto a chassis or surface of assay consumable storage assembly (103) at ornear a plane that includes the center of mass of assay assembly (103).

The eccentrics (115, 131 and 133) are cylindrical components positionedabout the rotating shafts (113, 123 and 125, respectively) having aninner and outer diameter (125 and 137, respectively) that do not sharethe same centerline. The rotating shafts are received within the innerdiameter of the eccentrics, and the eccentrics are received within ballbearing receivers on horizontal orbiting platform (102), which supportsthe assay consumable storage assembly (103) as best shown in FIG. 10(a),and/or within ball bearing receiver adapted to receive top eccentric(115). The distance between the centerlines of the inside and outsidediameters of the eccentric determines the orbital radius of theapparatus. For example, in the embodiment shown in FIG. 10(c), thedistance between the centerlines of the inside and outside diameters is2 mm; therefore the orbit radius is 2 mm, but this configuration can beadjusted without departing from the spirit or scope of the invention. Inone embodiment, all rotating components (e.g., the motor, drive shafts,and counterweights) rotate at the same speed and in the same direction.

In another embodiment, at least two bottom eccentrics (131 and 133) areattached to the horizontal orbiting platform (102) to minimize orpreferably prevent the assay consumable storage assembly (103) fromrotating about a single rotating axle. Preferably top eccentric (115) isused to minimize or prevent the shaft (113) from orbiting—shaft (113)should primarily rotate or only rotate. Additional bottom and topeccentrics can be used. Similarly sized eccentrics are used on thebottom mounting plate and on top of shaft (113) to mechanicallyconstrain the shaft vertically, in other words to help ensure that theentire assay consumable storage assembly (103) orbits uniformly aboutthe vertical Z axis. All the eccentrics are preferably rotating in-phasewith each other to minimize vibration. Preferably, shafts (113, 131 and133), which connect the eccentrics to the drive pulleys rotated by belt(127), have single rotational axes.

In yet another embodiment, the eccentrics rotate and the storageassembly orbits but preferably does not rotate. The rotational positionof the eccentrics about their shaft axis corresponds to the orbitalposition of the storage assembly about its central axis. The topeccentric (115) is preferably positioned to be about 180° out-of-phasewith the rotating counter-weight (105). Counter-weight (105), which isprovided to minimize the undesirable turbulence or the tendency to“walk” as best shown in FIG. 10(c), has an adjustable component 106which can be moved toward or away from rotating shaft (113) to increaseor decrease the angular momentum of the counter-weight. In onenon-limiting example, the mass of the assay consumable storage assembly(103) is about 5,000 grams and the mass of the counterweight (105) isabout 412 grams.

The system spring constant (k) of the assay consumable storage assembly(103) is preferably substantially high, so that the resonant frequency

${f = {\frac{1}{2\pi}\sqrt{\frac{k}{m}}}},$

where k is the system's spring constant and m is the system's mass, issubstantially high. Preferably, the assay assembly (103)'s resonant ornatural frequency is above the rotating frequency of the orbital shakerassembly (101). Preferably, the assay consumable storage assembly (103)contains no spring or damper.

Detailed views of a shelving subassembly (104) are shown in FIGS.11(a)-(c), The shelving assembly includes a housing (201) having a top(202), a back (203), left and right housing walls, which can be doublewalls, (204 and 205, respectively), and a plurality of sets ofvertically aligned storage units. In FIGS. 11(a)-(b) two sets ofvertically aligned storage units are shown (206 and 207, respectively).Storage units within a set are aligned or stacked, (e.g., 208-209) andeach storage unit includes an introduction aperture (210) and a doorconfigured to seal the aperture (211).

The shelving subassembly comprises an array of sets of verticallyaligned storage units. The array can be rectilinear, circular, orpolygonal. In one embodiment, the array is an M×N rectilinear array ofsets of vertically aligned storage units, wherein M and N are integers.One embodiment of a rectilinear array is shown in FIGS. 11(a)-(b) whichincludes two sets of storage units (206 and 207, respectively) adjacentto one another in the subassembly forming a 2×1 array. Alternativeconfigurations of a rectilinear array are shown in FIGS. 11(d)-(f),which depict a 2×2 array (2(d)), 3×3 array (2(e)), and a 4×4 array(2(f). In addition, the array can be polygonal or circular, as shown inFIGS. 11(g)-(i). If the array is polygonal, it is a regular polygon,e.g., a triangle, pentagon, hexagon, heptagon, octagon, nonagon,decagon, or a dodecagon, as shown in FIGS. 11(g) and 11(h), where X isan integer from 1-7. Alternatively, the array is circular, as shown inFIG. 11(i). In the embodiments shown in FIGS. 11(g)-(i), the arraycomprises 360°/P sets of storage units, wherein P is an integer and thesets of storage units are positioned within the shelving subassemblyabout a central axis (212-214, respectively).

Each shelving subassembly can include up to one hundred individualstorage units, preferably up to forty individual storage units, and morepreferably, up to twenty-four individual storage units. The skilledartisan will readily appreciate that numerous arrangements of storageunit sets in a shelving subassembly can be configured, varying in thenumber of sets as well as the number of vertically aligned storage unitsin a given set or collection of sets, as long as the apparatus includesa sufficient counterweight positioned within the storage assembly at aheight corresponding to the resultant center of mass of the storageassembly and the orbiting platform. In a specific embodiment, eachadjacent set of storage units sharing an adjoining wail (215; e.g., 206and 207) comprise the same number of storage units.

As shown in FIGS. 12(a)-(b), the apparatus can include two or morecounterweights, where the multiple counterweights are distributed evenlyso that the resulting center of mass of the multiple counterweightscoincides with the resultant center of mass of the orbiting components.As described above, a single counterweight would be positioned tocoincide with the center of mass of the orbiting components. In theembodiment shown in FIG. 12(a), the two or more counterweights (301 and302, respectively) are in operative communication with one rotating axle(303). Preferably, counterweights (301, 302) are located symmetricallyabove and below a centroid plane of the system shown in FIG. 12(b).Alternatively, as shown in FIG. 12(b), a first counterweight (304) is inoperative communication with a corresponding first rotating axle (305)and a second counterweight (306) is in operative communication with acorresponding second rotating axle (307), wherein each axle is driven bya timing belt (308) such that each rotating axle is driven in unison bythe orbital shaker assembly. Preferably, counterweights (304, 306) arelocated at or near a centroid plane of the orbiting system shown in FIG.12(b).

Any suitable orbital shaking mechanism can be used in the apparatus. Asdescribed in U.S. Pat. No. 5,558,437, the disclosure of which isincorporated herein by reference, conventional shaking mechanisms candrive the platform in an orbital translation and include one or morevertical shafts driven by a motor with an offset or crank on the upperend of an uppermost shaft such that the axis of the upper shaft moves ina circle with a radius determined by the offset in the shaft, i.e., bythe crank throw. The upper shaft or shafts are connected to theunderside of the platform via a bearing to disconnect the rotationalmovement between the upper shaft or shafts and the platform. Onmulti-shaft mechanisms, rotation of the platform is generally preventedby a four-bar-link arrangement of the shafts. On single shaftmechanisms, the rotation of the platform is generally prevented byconnecting an additional linkage or a compliant linkage between theplatform and base.

As described above, each storage unit is sized to accommodate an assayconsumable, e.g., a microlitre plate, and includes a latching mechanismto secure the consumable within the storage unit. An exemplary platelatching mechanism is shown in FIGS. 13(a)-(d) which is configured toreceive and engage an exemplary plate placed on the storage unitplatform (401) (or a consumable having the same footprint and externalphysical geometry as a multi-well/microtitre plate configured for use inan apparatus as described herein). The plate has at least a first,second, third, and fourth sides, wherein the first and third sides aresubstantially parallel to each other and the second and fourth sides aresubstantially parallel to each other. The outside edges of the platefollow a standard design convention for multi-well/microtitre plates andinclude a skirt (402) that surrounds and is at a height lower than thewalls of the plate (an enlarged view is shown in FIG. 13(b)). The platelatching mechanism is designed to push the outside edge of the skirt ontwo orthogonal sides of the plate against two corresponding physicalstops in the plate platform, to apply a downward physical force indefined locations on the top of the plate skirt to reproducibly andfixedly hold the plate.

In the embodiment shown in FIG. 13(a), the plate latching mechanism(403) is perpendicular to the platform edge aligned with the plateintroduction aperture of the storage unit (404). The plate latchingmechanism comprises a latching member (405) biased to the clampingposition and consisting of two pedals (406 and 407, respectively). Twocleats (408 and 409, respectively) located on the same side as pedals(406, 407) and two cleats (421, 422) located on the opposite sideconfigured to vertically constrain the plate skirt. Referring to FIG.13(c), the first pedal (406) is adapted to push the first side of themulti-well plate toward the first cleat (408) which engages with theplate skirt (402). The first cleat (408) engages with the plate skirtand provides a hard mechanical limit to the vertical movement of theplate. As the plate is pushed toward the inside of the platform, thesecond cleat (409) engages with the plate skirt and further restrainsvertical movement of the skirt of the plate. As shown in FIG. 13(d),when the plate is fully inserted on the platform and the latchingmechanism is completely engaged, first and second cleats (408, 409)along with opposite third and fourth cleats (421, 422) engage the plateskirt and limit the plate's vertical movement. Pedal (407) provides alateral bias to the plate and pedal (406) provides both a lateral andrearward bias to the plate. In the embodiment shown in FIG. 13(d), theplate is pushed against the rear end of the plate platform (410),opposite the plate introduction aperture, as well as the side of theplatform opposite the latching mechanism (411).

According to another aspect of the present invention, an optional airflow path (425) is provided internal to assay consumable storageassembly (103), as best shown in FIGS. 14(a) and 14(b). This air flowpath (425) comprises a number of vertical air shafts (427)interconnecting with a number of horizontal air shafts (429) betweenhorizontal orbiting platform (102) and the lower shelving subassembly(104) to allow cooling air to flow through or to circulate through assayconsumable storage assembly (103), and preferably between verticallyaligned storage units (106-109). An air exhaust or blower assembly (431)is provided to pull air through this gap. Alternatively, the air exhaustor blower assembly (431) may push air into air flow path (425). Anotheroptional air shall may be provided in the space between the uppershelving subassembly 104 and the lower shelving subassembly 104.

FIG. 14(c) illustrates another side view of shaker/incubator 28, whichincludes assay consumable storage assembly 103 and which shows thedirections of airflow through horizontal shafts 429 and verticalairshafts 427, as shown. The cooler air is pulled from the enclosurewithin upper compartment 14 of housing 12 by blower assembly 431preferably through the horizontal shaft 429 between lower shelvingsubassembly 104 and orbiting platform 102, as shown, into assayconsumable storage assembly 103 to cool the assembly. Heat may begenerated by the rotational equipment in orbital shaker 101 or by assayconsumables 111 stored within assay consumable storage assembly 103.Preferably, a temperature sensor 435, which can be a thermistor ispositioned at an inlet to either a horizontal shaft or a vertical shaft,is positioned to measure and to control the temperature of upper chamber14, as discussed further below. Preferably, temperature sensor 435 ispositioned in front of horizontal airshaft 429 between lower shelfassembly 104 and horizontal orbiting platform 102, as shown in FIG.14(c).

Warm exhaust air by blower assembly 431, labeled by arrow 437 in FIGS.14(c), is ducted into hood 439 which is connected to chimney 441, asshown in partial rear view of FIG. 14(d). Warm exhaust air 437 pulledfrom assay consumable storage assembly 103 and ducted through hood 439and chimney 441 is brought to the inlets of coolers 38, as best shown inthe partial front view of FIG. 14(e). The warm exhaust air is cooled bycoolers 38 and is returned to upper chamber 14 of housing 12 via plenum443 to the upper part of upper chamber 14 and through inclined flowdiverted 68, which is previously illustrated in FIG. 8. Plenum 443 is analternative to top flow plenum 62, discussed above and shown in FIG. 7,and is shorter in length and directs the cooled air downward into thecenter of upper chamber 14, as shown in FIG. 14(e). Plenum 443 is usedinstead of plenum 62 when there is insufficient room at the front ofupper chamber 14 for the cooling air to flow to the front and. reversecourse backward toward the inlet of coolers 38.

Temperature sensor or thermistor 435 is advantageously connected to atleast one or more coolers 38 and acts as its/their thermostat, i.e., toset a temperature that the one or more coolers 38 must work to maintainthe pre-set or pre-select temperature at the position of thermistor 435.In other words, the one or more coolers 38 have to work until thetemperature of the air entering assay consumable storage assembly 103 attemperature sensor 435 reaches the pre-set temperature to ensure thatthis air is sufficiently cool to remove heat generated withinshaker/incubator 28 including assay consumable storage assembly 103.Thermistor 435 acts as a thermostat for the shaker/incubator and assayconsumable storage assembly 103.

Thermistor 435 when connected to at least one cooler 38 acting as athermostat can be applied to any heat generating device within housing12, including but not limited to assay consumable storage assembly 103.In an alternative, warm exhaust air 437 may be exhausted outside ofhousing 12 to reduce the heat load on coolers 38 with appropriatefilters.

EXAMPLE 1

System 10 is designed for ultra-high throughput testing of clinicalsamples with ECL detection technologies, such as those from Meso ScaleDiagnostics (“Meso Scale”) of Rockville, Md. To achieve high throughput,system 10 uses multiplexed testing in a 96-well plate format and canprocess batches of up to 20 plates. It has a central robot arm 26 thattransfers plates between components that perform different assay steps:a 96 channel pipettor 32, barcode reader, plate washer 30, plate shaker28 and a plate ECL reader 36 from Meso Scale. System 10 is afree-standing fully automated system. System 10 is functional and betaunits are in use at Meso Scale.

Key features of the system 10's platform include, but not limited to:

-   -   1. Batch processing of up to 20 assay plates (1600 samples per        batch assuming 80 samples per plate (singlicate)+16 wells used        for calibrators or controls)    -   2. Throughputs as high as 12,800 samples per day (8 batches of        20 plates per day)    -   3. All pipetting and washing steps are performed using        96-channel components capable of processing all the wells in a        plate simultaneously    -   4. Assay processing is carried out in a temperature controlled        enclosure and all steps are precisely timed to provide highly        reproducible and precise results    -   5. A custom designed shaker (described in commonly owned        provisional application Ser. No. 62/143,557, incorporated herein        in its entirety) with a 20 plate capacity provides rapid        antibody binding kinetics; each plate is incubated in a separate        enclosed chamber within the shaker to prevent evaporation    -   6. Ability to carry out samples dilutions    -   7. Simple scheduling approach does not require interleaving of        assay operations.

System 10 uses individual off-the-shelf and custom built components thatfunction together as one fully automated system. The off-the-shelfcomponents include a MESO QuickPlex® SQ 120 Reader (available from MesoScale), a BioTek® 96-channel plate washer, a Precise Automation platehandling robot, a barcode reader and an Apricot Designs™ 96-channelpipetting head, The custom built components of the platform include aplate hotel/shelves, a 20-plate shaking incubator, two pipette tipwashing manifolds, a four-plate pipetting deck, and a pipettor gantry tosupport the pipetting head. The deck and all components are locatedwithin an enclosure with heating/cooling units that maintain theenclosure at a set temperature (FIGS. 2 and 3).

The platform attains ultra-high throughput by processing entire 96-wellplates at once using 96-channel components. This approach allows theprocessing operations to be divided into a series of processing cyclesseparated by incubation periods, where the time associated with anyspecific processing cycle is a discrete amount of time, for example,less than 3 minutes. It is, therefore, possible to schedule each cycleon each plate to be carried out in 3 minute intervals, simplifying thescheduling of operations while maintaining tight control over the timingof each cycle. By separating each assay cycle with binding reactionincubation times of one hour, batches of plates, e.g., 20, can be runwhile maintaining the 3 minute intervals between plates over multipleassay cycles. The system can be expanded for even higher throughput.

By way of a non-limiting example, in the case of a biodosimetry test(see Example 2), system 10 runs a single incubation assay with thefollowing automated assay processing steps, divided into cycles:

1, Sample addition cycle:

-   -   a. Robot removes source plate (with samples), reagent reservoir        (with detection antibody solution) and assay plate (MSD®        Multi-Array plate with capture antibody array from Meso Scale)        from plate hotel/shelves and places on the deck    -   b. Robot lifts lid from source plate to allow pipettor access    -   c. Pipettor transfers sample and detection antibody to wells of        assay plate    -   d. Robot transfers assay plate to shaker for one hour incubation    -   e. Pipette tips are washed using tip washing manifolds

2. Plate read cycle (scheduled one hour after step 1):

-   -   a. Robot removes assay plate from shaker and places on washer    -   b. Plate washer washes wells three times with wash buffer to        remove sample    -   c. Robot moves assay plate and reagent reservoir with read        buffer to deck    -   d. Pipettor transfers read buffer to wells of assay plate    -   e. Robot transfers plate to MSD SQ120 plate reader for analysis

By maintaining the 3 minute interval between plates, it is possible toprocess 20 plates in a batch with a time-to-first result of 1 hour and atime-to-final result of 2 hours, while maintaining strict control of thetiming of each cycle. Optionally, the binding of sample and detectionantibody may be separated into two separate cycles with separate 1 hourincubations to achieve optimal assay performance. In this case, thetime-to-first result would be 2 hours and the time-to-final result wouldbe 3 hours for a 20 plate batch.

To prepare the system and reagents to run the biodosimetry test on abatch of 20 plates, the operator would follow the process describedbelow:

-   -   Lyophilized detection antibody is rehydrated and transferred to        a reagent reservoir    -   Read buffer, supplied as a liquid bulk reagent, is added to a        second reagent reservoir    -   Lyophilized calibrators and controls (supplied with kit in tubes        with the kit) are rehydrated    -   Samples, controls and calibrators are transferred from tubes        into 96-well source plates    -   Columns 1 and 2 on the plates are reserved for a 7-point        calibration curve run in duplicate and 2 controls; columns 3 to        12 are used for 80 samples)    -   This step can be performed manually or, for higher throughput,        with automated sample transfer workstations that are found in        most clinical laboratories    -   The user logs into the system using his or her login credentials    -   The user selects the assay type (Biodosimetry Test in this        case); this defines the assay protocol setup including the        dispense volumes, incubation times, etc.    -   Following graphical diagrams provided by the software, the user        adds the MSD assay plates, source plates and reagent reservoirs        to the plate rack    -   The system runs a setup routine that takes inventory of all        source plates (barcodes are read) and reagent reservoirs to        confirm the location of all components; the system also confirms        with the user that the bulk reagents have been replaced    -   The system executes the automated Biodosimetry assay protocol        (described above)    -   Used assay plates and reservoirs are removed from the plate        hotel/shelves    -   Results are calculated by the software and displayed on the        touchscreen GUI

System 10 can process 20 plates within 2 hours. For highest throughput,the next set of 20 sample source plates (containing sample, calibratorsand controls) can be prepared while the current set of 20 plates isrunning. The source plates can be prepared manually, however commercialoff-the-shelf systems for sample reformatting can be used to moreefficiently complete this task and maintain the same throughput assystem 10. Automated systems found in most large clinical laboratoriescan centrifuge blood tubes, de-cap tubes, and pipet plasma samples intoa pre-defined layout into system 10's sample source plates. Thesesystems can also be programmed to transfer calibrators and controls tothe sample source plates, or the user can perform this task manuallyonce the samples have been processed and added to the source plates. Thesoftware for system would have the capability of communicating withthese automated systems to upload the locations of each sample(identified by a unique barcode ID) within each source plate (alsoidentifiable by a unique barcode ID).

EXAMPLE 2: Biodosimetry Assay

Detailed descriptions of biodosimetry assays and algorithms that can becarried out using an instrument and/or methods in accordance with theinvention are described in U.S. application Ser. No. 14/348,275, (U.S.Publication No. 2014/0315742) which is incorporated by reference hereinin its entirety. Included in the application is the use of a panel ofsix radiation biomarkers in plasma or blood (Flt-3L, CD20, CD177; TPO,LBP, salivary amylase) to estimate the dose of exposure of an individualthat may have been exposed to radiation. This specific panel isdescribed for illustrative purposes. The invention encompasses use ofthe instrumentation and methods described herein to conduct assays forany one of these biomarkers whether alone or in combination with otheranalytes, or any combination of two, three, four, or five of thesebiomarkers, with or without other analytes contained in the same assaypanel. Specifications for such a test on System 10 are outlined below:

TABLE Specifications for the Biodosimetry Test Conducted on System 10.Specification Details General Specifications Number of biomarkers 6biomarkers System 10 supports 25-plex measurements 2 internalBiodosimetry test has 6 biomarkers (Flt-3L, controls CD20, CD177, TPO,LBP, salivary amylase) and 2 internal procedural controls Sample typePlasma K2EDTA plasma from a venous draw Sample volume 50 μl Time toresult (20 plate 1 hr (1st result) Assuming single 1 hour incubationbatch; 1,600 samples) 2 hr (final result) Throughput 12,800 samplesAssuming 8 batches of 20 plates per day, 80 (samples/day) samples perplate run in singlicate, and 16 wells used for calibrators or controlsResult reporting Visual and Visual: results on touch screen interfaceelectronic Electronic: results stored on system and available throughnetwork interface Patient tracking Patient ID Patient ID will beelectronically linked to test linked to results results in records;barcodes can be used for patient IDs Internal procedural Yes Negativeand positive internal procedural controls controls based on artificialantigens External QC controls Yes Positive control (mimics moderatedose) Negative control (mimics no exposure) Calibration of biomarker YesEach plate is independently calibrated using a assays set of calibratorscontaining all 6 biomarkers Performance of Biodosimetry Test Measurabledose range 0.5-10 Gy Assesses dose over wide dose range Time window fortesting 24 hrs-7 days Dose assessment up to 20 days post-exposure (timeof sample post-exposure may be possible collection) Dose accuracy: ±0.5Gy Quantitative assessment of dose Doses <2 Gy ±25% Doses ≥2 Gy Clinicalsensitivity 99% For patients receiving doses ≥2 Gy Clinical specificity97% For unexposed individuals Performance of Individual Biomarker AssaysAssay precision ±10% (15%) Intra-run (inter-run) coefficients ofvariation Assay linearity ±20% Linearity of biomarker quantitation inrelevant concentration range Instrument Properties Temperature - ambient20° C. to 26° C. Allowable environmental temperature range Temperature -assay 23° C. ± 1° C. Temperature variation inside System 10 Size Fits onpallet 5′ × 3′ × 5′ (W × D × H) Power Standard 120 or 208-240 VAC208/240 VAC Ramp-up time ≤1 hour Time to reach internal temperature setpoint Uptime/maintenance 23 hrs/day Maintenance includes replenishingbuffers, uptime emptying waste, and performing cleaning ≤1 hr cycles onthe plate washer maintenance Ease of use/Test CLIA Fully automatedlaboratory instrament with bar- category moderately coded sampletracking complex Test Consumables Test kit components Assay MaterialsEach test kit contains: required to run 20 assay plates (96-wellMULTI-ARRAY) 20 plates Detection reagent (lyophilized) Calibrators(lyophilized) Assay diluent (dry) External controls (lyophilized)Package insert Kit shelf life ≥3 years Shelf life is for consumablesstored at 4° C.; all biological reagents will be in a dry format formaximum reagent stability and long shelf life Bulk Consumables andAdditional Sample source plates Fluids consumables Pipette tips andfluids Reagent reservoirs needed to run Wash buffer system Read bufferDI water Plate lids Bulk fluid shelf life ≥3 years Room temperaturestorage

Assay Formats That Can Be Run on System 10

System 10 can be configured to run any number of assay formats bymodification of the processing cycles (as described above) used toprocess assay plates. Several illustrative examples are provided belowfor different immunoassay formats, although the basic approaches areclearly applicable to other assay types including binding assays usingnon-antibody based binding reagents (e.g., nucleic acid hybridizationassays). System 10 is specifically designed for carrying out assaysusing ECL detection and Meso Scale (MSD) MULTI-ARRAY® assay plates, butthe approaches are applicable to techniques using other multi-well plateconsumables and detection technologies.

The different assay formats are described through a table that lists theprocessing cycles used by System 10 to complete the assay process. Eachcycle may comprise one or more of the following assay steps: (i) usingthe robotic arm to select a target plate from the hotel or shaker andmoving the plate to the washer for a plate wash, (ii) using the roboticarm to move the target plate to the pipetting deck for pipettingoperations, (iii) and (iv) selection of up to two source/reagent platesincluding moving the plates from the hotel to the pipetting deck andusing the pipettor to transfer solutions to the target plate, (v)pipette tip wash (after each plate or only after the last plateundergoing a specified cycle), (vi) transfer of the target plate to anincubation location where incubation can be carried out with shaking(i.e., in the shaker) or without shaking (in the plate hotel) and (vii)transfer to the plate reader for carrying out the assay measurement. Thetable lists which steps are carried out in each cycle and identifies thetarget and source/reagent plates by content, where “capture”,“detection” and “sample” refer to source/reagent plates that containcapture reagents, detection reagents, or samples, respectively.

Two-Step Sandwich Immunoassay. In the two-step sandwich immunoassay, thewells of a MSD assay plate (with one capture antibody or an array ofcapture antibodies immobilized on the bottom of each well) are incubatedfirst with sample diluted in an assay diluent and then with labeleddetection antibodies prior to measurement of the labeled sandwichcomplexes that form. The protocol as shown includes a blocking cycle asthe first cycle; optionally, this cycle may be omitted. One simplevariation of this protocol includes an additional cycle (3 a) betweencycles 3 and 4. This protocol is used when the detection reagent incycle 3 does not comprise a label of the type detected in the reader,Cycle 3 a is like cycle 3 except that Source Plate 1 contains a labeledsecondary reagent that binds the detection reagent, with a labelappropriate for the reader. The use of a labeled secondary detectionreagent is well known in the art. Specific examples include the use oflabeled anti-species antibodies to detect antibody detection reagents,or the use of labeled streptavidin to detect biotin containing detectionreagents.

Target Source Source Cycle Description Wash Plate Plate 1 Plate 2 TipWash Incubation Read 1 Block Plate No MSD Blocker None After last Staticor No Assay plate Shaking 2 Add Yes MSD Sample Diluent After eachShaking No Sample Assay plate 3 Add Yes MSD Detection None After lastShaking No Detection Assay plate 4 Read Yes MSD Read None None No YesAssay buffer

Sandwich Assay Including Antibody Immobilization Step. This protocol issimilar to the sandwich assay described above except that instead ofusing an assay plate pre-coated with a capture antibody, the assay plateis either uncoated or coated with a generic capture reagent such asstreptavidin. The protocol, therefore, includes an additional cycleduring which the capture antibody is adsorbed onto the uncoated plate orcapture by binding to the capture reagent (for example, through thebinding of a biotin-labeled capture antibody to immobilized streptavidinin the well). As in the previous table, the blocking cycle may beomitted. The protocol as described in the table can be used toimmobilize a single capture reagent per well, or can be used for thesolution phase assembly of an array of capture reagents as described inUS Published Patent Application No. 20140256588. For example, the wellsin the assay plate may each have an immobilized array of differenttargeting reagents (e.g., oligonucleotides), and the capture reagent(i,e., the contents of Source Plate 1 in Cycle 1) may be a mixture ofdifferent capture reagents linked to different targeting reagentcomplements (e,g., oligonucleotides complementary to the targetingagents), such that when this mixture is incubated in a well, thetargeting reagents and their complements bind and the different capturereagents immobilize on different elements of the targeting reagent arrayto form a capture reagent array.

Target Source Source Cycle Description Wash Plate Plate 1 Plate 2 TipWash Incubation Read 1 Coat No MSD Capture None After last Shaking NoCapture Assay plate 2 Block Plate Yes MSD Blocker None After last Staticor No Assay plate Shaking 3 Add Sample Yes MSD Sample Assay After eachShaking No Assay Diluent plate 4 Add Yes MSD Detection None After lastShaking No Detection Assay plate 5 Read Yes MSD Read None None No YesAssay buffer

Bridging Immunogenicity/Serology Assay. In this protocol, antibodiesagainst a specific antigen or drug are identified by their ability tosimultaneously bind two copies of the antigen or drug to form a sandwichcomplex. In the embodiment described in the table a mixture(“mastermix”) of antigen linked to biotin (or some other bindingreagent) and antigen linked to a label detectable in the System 10 platereader (such as an ECL is aliquoted into a plate (the Mastermix plate).Sample and acid are transferred into a plate (the Treatment plate), soas to dissociate any antibody complexes that may be present in thesample. The acid-treated sample and a neutralization buffer are thencombined with mastermix in the mastermix plate and incubated to allowthe formation of sandwich complexes comprising biotin-antigen,antibody-of-interest, and labeled-antigen. The resulting solutions arethen transferred to an assay plate having wells comprising immobilizedstreptavidin (or an appropriate binding partner for the binding reagentlinked to the antigen) and incubated to capture the sandwich complex tothe immobilized streptavidin where it can be measured in the platereader. In some cases, it may be desired to run this protocol withoutacid dissociation, in Which case the acid in Source Plate 1 in Cycle 3may be replaced with a non-acidic dilution buffer and, optionally, theneutralization buffer may be omitted or replaced with an assay diluent.Alternatively, Cycle 3 may be omitted completely and sample may be addeddirectly to the Mastermix plate in Cycle 4 (i.e., Source Plate 2 is asample plate). Note that since the incubations after Cycles 1 and 2occur in the plate hotel, these incubations may continue in parallelwith the incubations of later cycles until the resulting plates arerequired in Cycles 4 and 5, respectively.

Target Source Source Tip Cycle Description Wash Plate Plate 1 Plate 2Wash Incubation Read 1 Aliquot No Mastermix Mastermix None After lastStatic No Mastermix Reservoir plate 2 Block Plate No MSD Blocker NoneAfter last Static No Assay plate 3 Acid No Treatment Acid Sample Aftereach Shaking No Dissociation Plate plate 4 Sample + No MastermixNeutralization Treatment After each Shaking No Mastermix Buffer Plateplate 5 Load Yes MSD Mastermix None After each Shaking No Assay PlateAssay plate 6 Read Yes MSD Read buffer None None No Yes Assay

Amplified Immunoassay. System 10 may be used to carry out binding assaysthat employ an amplification step to increase sensitivity. In theexample of a Two-Step Immunoassay described above, the process mayinclude an additional amplification cycle (3 a) between cycles 3 and 4to prepare for or carryout the amplification procedure. In the case thatthe detection reagent includes an enzyme label, the cycle 3 a couldinclude adding an enzyme substrate (in Source Plate 1) to the assayplate (the Target Plate) where conversion of the substrate by the enzymeleads is detectable by the reader. Alternatively, cycle 3 a could beomitted and the substrate could be added from Source Plate 1 or 2 incycle 4, just prior to transfer of the assay plate to the reader. In thecase that the detection reagent includes a nucleic acid label, cycle 3 acould include adding the reagents for amplifying the label from SourcePlates 1 and/or 2 (e.g., by PCR or isothermal nucleic acidamplification), the amplification being carried out in the subsequentincubation period. The table below describes the automated assay processfor carrying out an amplified binding assay as described US PublishedPatent Application No. 20140272939 in the context of an immunoassayusing antibodies as analyte binding reagents, although the process couldclearly be applied to assays using other types of binding reagents. Eachwell of the MSD Assay Plate has an immobilized capture antibody that is,optionally, co-immobilized with an anchoring reagent comprising ananchoring oligonucleotide sequence. The procedure includes an optionalblocking cycle (Cycle 1), followed by cycles for adding sample to bindanalyte to the capture antibody (Cycle 2) and for adding detectionreagent to bind to the captured analyte (Cycle 3). In this embodiment,the detection reagent is a mixture of a first detection antibody linkedto a. first nucleic acid probe and a. second detection antibody linkedto a second nucleic acid probe, both of which bind to captured analyteto form a complex on the well surface comprising the capture antibody,the analyte and both detection antibodies. In the ligation cycle (Cycle3), a ligation mixture is added to each well comprising a ligase and oneor more connector nucleic acid sequences that are linear sequences thatcomprise regions complementary to the first and second probes, so thatwhen incubated in the presence of one of the complexes of captureantibody, analyte and first and second detection antibodies, theconnector sequence(s) are ligated to form a circular nucleic acidsequence hybridized to the first and second probes in the complex. Ifthe optional anchoring oligonucleotide is included, the connectorsequence(s) include an anchoring region that matches a region of theanchor sequence (i.e., they both hybridize to the same complementarysequence). In the amplification cycle (Cycle 4), an amplificationmixture is added to each well comprising a DNA polymerase and a labeleddetection probe (comprising a detection sequence that matches adetection region in the connector sequence(s)). When the amplificationmixture is incubated in the presence of the circular nucleic acid boundto the first and second probes, the first probe is extended by rollingcircle amplification and the labeled detection probes bind to theextended product. The extended product also binds to the anchoringreagent, if present. In the read cycle, read buffer is added to thewells and labeled probe is detected in the reader to measure thepresence of the analyte.

Target Source Source Tip Cycle Description Wash Plate Plate 1 Plate 2Wash Incubation Read 1 Block Plate No MSD Blocker None After last Staticor No Assay plate Shaking 2 Add Sample Yes MSD Sample Diluent After eachShaking No Assay plate 3 Add Detection Yes MSD Detection None After eachShaking No Assay Ab mixture plate 4 Ligation Yes MSD Ligation None Afterlast Shaking No Assay mixture plate 5 Amplification Yes MSDAmplification None After last Shaking No & Detection Assay mixture plate6 Read Yes MSD Read buffer None None No Yes Assay

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theclaims. Various publications are cited herein, the disclosures of whichare incorporated by reference in their entireties.

1. An ECL immunoassay system comprising a housing that encloses apipette dispenser, a plurality of multi-well plates adapted to hold ECLcomplexes attached to electrodes contained in the plurality ofmulti-well plates, which are sized and dimensioned to be stored in anincubator, a blower, an ECL reader and at least one cooler, wherein theat least one cooler is located proximate to a back surface of thehousing, wherein the incubator comprises at least one airflow channeldisposed therewithin and the blower is positioned to pull air from thehousing through the at least one airflow channel, and said air is ductedto an inlet of the at least one cooler to cool said air.
 2. The ECLimmunoassay system of claim 1, wherein said air is blown by the at leastone cooler through a plenum into the housing.
 3. The ECL immunoassaysystem of claim 1, wherein a temperature sensor connected to the atleast at least one cooler is positioned proximate to an entrance of theat least one airflow channel.
 4. The ECL immunoassay system of claim 3,wherein said temperature sensor is a thermostat of the at least onecooler.
 5. The ECL immunoassay system of claim 3, wherein saidtemperature sensor comprises a thermistor.
 6. The ECL immunoassay systemof claim 1, wherein said air is ducted to an inlet of the at least onecooler through a hood positioned above the blower.
 7. The ECLimmunoassay system of claim 6, wherein said air is further ducted to aninlet of the at least one cooler through a chimney connected to said ahood,
 8. The ECL immunoassay system of claim 1, wherein the at least onecooler comprises at least one thermoelectric cooler.
 9. The ECLimmunoassay system of claim 1, wherein the at least one airflow channelcomprises a substantially horizontal airflow channel.
 10. The ECLimmunoassay system of claim 1, wherein the at least one airflow channelcomprises a substantially vertical airflow channel.